Biocontrol of weeds

ABSTRACT

The present invention is directed to a biocontrol agent  Plectosporium tabacinum  and methods for the biocontrol of weeds using the biocontrol agent. Preferably the weeds are cleavers ( Galium aparine  L and  Galium spurium  L.), and the biocontrol agent is  Plectosporium tabacinum  CL98-103 (ATCC deposit PTA-3463). The biocontrol agent is effective against herbicide-resistant and herbicide-susceptible cleavers, and it may be used in conjunction with other herbicides.

RELATED APPLICATION

[0001] The present invention claims priority from Canadian ApplicationNo. 2,324,215, filed Nov. 6, 2000, which application is incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the biocontrol of weed growthusing a bioherbicide. More specifically, the present applicationpertains to the use of a fungal bioherbicide for the control ofcleavers.

[0003] BACKGROUND OF THE INVENTION

[0004] Cleavers [false cleavers (Galium spurium L.) and cleavers (G.aparine L.)] are weeds of important economic impact in western Canada,especially for producers of canola (Brassica napus L. and B. rapa L.)(Malik and Vanden Born, 1988). Weed surveys in the prairie provinces ofCanada have indicated that cleavers populations have increased duringthe past 10 years and their abundance ranking have increased morerapidly than other cropland weeds (Thomas, 1998). Heavy infestations ofcleavers in canola cause severe yield losses, up to 18% with 100 falsecleavers plants/m² through crop/weed competition Malik and Vanden Born,1987). Another problem with cleavers in canola is that cleavers seedsare similar in shape and size to canola seeds, making mechanical seedseparation difficult. Cleavers seed contamination in canola leads todowngrading of canola quality, has implications for the crushingindustry, and contributes to the spread of weed infestations. Underlegislation in the Canada Seeds Act, no cleavers seed is allowed inpedigreed canola seed and thus pedigreed seed producers of canola cannottolerate land infested with these weeds. Cleavers are not only difficultto control in canola but are an increasing problem in other major cropsof Western Canada such as spring wheat (Triticum aestivum L.), barley(Hordeum vulgare L.), and pea (Pisum sativum L.).

[0005] Considerable efforts have been made to find effect herbicidecontrols for cleavers over the past decade. Chemical herbicides used forcleavers control include several acetolactate synthase (ALS) inhibitorsalong with auxin-type herbicide combinations. Unfortunately, herbicideresistance has been detected in populations of false cleavers (Hall etal., 1998). This herbicide-resistant false cleavers biotype showscross-resistance to quinclorac and ALS inhibitors, includingimazethapyr, one of the products for which herbicide-tolerant canola hasbeen developed. With continuing herbicide use and herbicide-tolerantcanola cultivation (approximately 20-40% or more of the canola acreagein Canada), herbicide resistance may become more common in falsecleavers.

[0006] To date, no bioherbicides are available for control of cleavers.Thus, there is a need in the art for new or alternative cleavers controlstrategies. There is also a need in the art for cleaver controlstrategies for both conventional and herbicide tolerant (HT) canola.Further there is a need for biological control agents that complementherbicide use by introducing novel modes of action to mitigate herbicideresistance development and to provide a component within an integratedpest management system.

[0007] It is an object of the invention to overcome disadvantages of theprior art.

[0008] The above object is met by the combinations of features of themain claims, the sub-claims disclose further advantageous embodiments ofthe invention.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the biocontrol of weed growthusing a fungal bioherbicide.

[0010] According to the present invention there is provided a biocontrolagent comprising Plectosporium. tabacinum CL98-103. Preferably, thebiocontrol agent comprisies

[0011] Plectosporium tabacinum CL98-103 deposit number PTA-3463 (ATCC).

[0012] Also according to the present invention, there is provided acomposition comprising the biocontrol agent (P. tabacinum CL98-103) anda carrier. Any carrier that permits the biocontrol agent to be deliveredto a target plant in a manner such that the biocontrol agent remainsviable and pathogenic may be employed in the composition. Examples ofcarriers include, but are not limited to clay, alginate, diatomaceousearth, growth medium, or a combination thereof. The growth medium maycomprise solid growth medium or liquid growth medium or a combinationthereof. Solid growth medium may comprise potato dextrose agar,Czapek-Dox agar, lima bean agar, V-8 juice agar, oatmeal agar, trypticsoy agar, dextrose tryptone agar, Cooke rose bengal agar, prune agar,malt extract agar, synthetic nutrient poor agar, Sabouraud dextroseagar, water agar, cornmeal agar or a combination thereof. Liquid growthmedia may comprise V-8 juice medium, Modified Richard's solution (MSR),Yeast extract broth (YEB), Richard's solution (RS), Czapek-Dox broth(CDB), Trichoderma medium (TM), Tryptic soy broth (TSB), Potato dextrosebroth (PDB), Nutrient broth (NB), Colletotrichum truncatum medium (CTM),Malt extract broth (MEB) or a combination thereof.

[0013] Also according to the present invention, there is provided amethod for controlling weeds, herbicide-resistant andherbicide-susceptible cleavers, herbicide-resistant andherbicide-susceptible false cleavers, and other weeds by infecting themwith the biocontrol agent defined above. The biocontrol agent may beadministered to the cleavers in combination with a herbicide.Preferably, the biocontrol agent is administered to the weeds at aboutthe one whorl stage or earlier.

[0014] Further according to the present invention as defined above, thebiocontrol agent or composition comprising the biocontrol agent mayadditionally comprise a surfactant. Preferably the surfactant is SilwetL-77, and is present in an amount of about 0.05% to about 0.1% byvolume.

[0015] Also according to the present invention as defined above, thereis provided a composition comprising spores of P. tabacinum CL98-103 anda carrier.

[0016] Further, according to the present invention there is provided amethod for the biocontrol of a weed plant under non-aquatic conditions,or conditions that do not requiring periodic submersion, usingPlectosporium. tabacinum as a biocontrol agent.

[0017] Also according to the present invention, there is provided amethod for growing and producing spores of Plectosporium. tabacinumCL98-103, comprising growing the fungal biocontrol agent or sporesthereof in a suitable liquid medium.

[0018] This summary of the invention does not necessarily describe allnecessary features of the invention but that the invention may alsoreside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] These and other features of the invention will become moreapparent from the following description in which reference is made tothe appended drawings wherein:

[0020]FIG. 1 shows characteristics of biocontrol agent CL98-103. FIG. 1Ashows spread cultures of biocontrol agent CL98-103 grown on PDA withcontinuous light. FIG. 1B shows single spore cultures of biocontrolagent CL98-103 grown on PDA with continuous light. FIG. 1C showsconidiophores of biocontrol agent CL98-103. Figure ID shows a condium ofbiocontrol agent CL98-103. FIG. 1E shows two conidia of biocontrol agentCL98-103.

[0021] FIGS. 1F-H show germinated conidia of biocontrol agent CL98-103.

[0022]FIG. 2 shows the disease reaction of false cleavers caused byfungal biocontrol agent CL98-103. FIG. 2A shows false cleavers at thecotyledon stage that have been treated with fungal biocontrol agent(right) or untreated (left).

[0023]FIG. 2B shows false cleavers at the 1-whorl stage that have beentreated with fungal biocontrol agent (right) or untreated (left).

[0024]FIG. 3 shows the effect of inoculum concentration and dew periodduration on disease development caused by Plectosporium tabacinumCl-98-103 on false cleavers, expressed as percent mortality (FIG. 3A)and reduction in dry weight (FIG. 3B) seven days after inoculation.False cleavers seedlings at the 1-whorl stage were inoculated withconidial suspension at a concentration of 5×10⁶, 1×10⁷, 5×10⁷, or 1×10 ⁸conidia/ml in 1% gelatin solution. Dew temperature was 22° C. (in dark)and plants were placed in the dew chamber for 9, 12, 16, or 20 h andthen returned to the greenhouse. Data represent the mean of threereplicates.

[0025]FIG. 4 shows the effect of Plectosporium tabacinum CL98-103inoculum concentration and plant growth stage on the control of falsecleavers, expressed as percent mortality (FIG. 4A) and reduction in dryweight (FIG. 4B) seven days after inoculation. Seedlings of falsecleavers at the cotyledon, and 1-, 2-, 3-, 4- or 5-whorl growth stage offalse cleavers were inoculated with a conidial suspension atconcentrations of 1×10⁸, 1×10⁷, 1×10⁶, 1¹⁰ ⁵ or 0 spores/ml in 1%gelatin solution. After inoculation, pots with inoculated seedlings wereplaced in a dark dew chamber for 24 h. Data represent the mean of threereplicates.

[0026]FIG. 5 shows the effect of dew period on disease developmentcaused by Plectosporium tabacinum CL98-103 on false cleavers, expressedas percent mortality (FIG. 5A) and reduction in dry weight (FIG. 5B)seven days after inoculation. Seedlings of false cleavers at the 1-whorlgrowth stage were inoculated with a conidial suspension at aconcentration of 1×10⁷ conidia/ml in 1% gelatin solution. Dewtemperature was 22°C. (in dark). Data represent the mean of threereplicates.

[0027]FIG. 6 shows the effect of temperature on radial mycelial growth(FIG. 6A) and spore production (FIG. 6B) of Plectosporium tabacinumCL98-103 on potato dextrose agar (PDA), Czapek Dox agar (CDA), lima beanagar (LBA) and V-8 juice agar (VA). The number of conidia per plate wasdetermined after 21 d incubation while mycelial growth was measuredafter 14 d incubation. Results of two trials with three replicates pertrial were combined for each medium.

[0028] Standard errors are indicated by vertical bars.

[0029]FIG. 7 shows the effect of pH on spore production of PlectosporiumtabacinumCL98-103 on standard agar media (potato dextrose agar; FIG. 7A)and in submerged liquid culture using Richard's solution (filledsquares), modified Richard's solution (filled circles), potato dextrosebroth (filled triangles), and yeast extract broth (filled diamonds; FIG.7B). On standard agar media, the number of spores per plate wasdetermined after 21 d incubation. For submerged liquid culture, thenumber of spores per ml was determined after 3 d incubation. Results oftwo trials with three replicates per trial were combined for eachmedium. Standard errors are indicated by vertical bars.

[0030]FIG. 8 shows the effect of carbon-to-nitrogen ratio on sporeproduction of Plectosporium tabacinum CL98-103 in a basal salt medium ofRichard's solution. Media with three carbon (sucrose) and nitrogen(KNO₃) concentrations and six different ratios of carbon to nitrogen(C:N) was prepared. Media with sucrose concentrations of 8.4 (FIG. 8A),21 (FIG. 8B), and 33.6 (FIG. 8C) gL⁻¹ and C:N rations of 40:1, 20:1,15:1, 10:1, 7.5:1, and 5:1 were prepared. Spore production in unmodifiedRichard's solution (50 gL⁻¹ sucrose and a 15:1 C:N ratio) was includedas a control. The number of spores per ml was determined after 3 daysincubation on an orbit shaker at 150 rpm.

DESCRIPTION OF PREFERRED EMBODIMENT

[0031] The present invention relates to the biocontrol of weed growthusing a fungal bioherbicide.

[0032] The following description is of a preferred embodiment by way ofexample only and without limitation to the combination of featuresnecessary for carrying the invention into effect.

[0033] The present invention provides a fungal bioherbicide of thePlectosporium species. The present invention also provides a method tocontrol both herbicide-resistant and herbicide-susceptible cleavers(Galium aparine L. and G. spufium L.). The method comprises applying aneffective amount of P. tabacinum to the weeds.

[0034] By “biocontrol agent” or “bioherbicide” it is meant an organism,typically a plant pathogen, that reduces the growth rate, development,or both the growth rate and development (as evidenced by reduced dryweight), possibly leading to death, of at least one target plantspecies. Preferably, the biocontrol agent exhibits selective activity(host specificity) when exposed to one or more plants, so that a plantof interest is not affected by the biocontrol agent, while one or moretarget plants, for example a weed species is susceptible to the effectsof the biocontrol agent. The present invention provides a fungalbioherbicide of the Plectosporium species. Preferably the fungus is a P.tabacinum strain (van Beyma) CL98-103. More preferably, the fungus is P.tabacinum deposited at the ATCC as PTA-3463, on Jun. 9, 2001.

[0035] By “plant of interest” it is meant the plant species for whichgrowth is desired when exposed to a biocontrol agent. Plants of interestmay include horticultural and agriculturally important species. Withoutwishing to be limiting, a plant of interest may be selected from thegroup consisting of canola (Brassica napus and B. rapa), spring wheat(Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativumL.), cauliflower (Brassica oleracea L.), oats (Avena sativa L.), alfalfa(Medicago sativa L.), lentil (Lens culinaris Medic.), flax (Linumusitatissimum L.), sunflower (Helianthus annuus L.), safflower(Carthamus tinctorius L.), potato (Solanum tuberosum L.), tomato(Lycopersicon esculentum L.), tobacco (Nicotiana tabacum L.), balsam(Impatiens balsami L.), celery (Apium graveolens L), parsnip (Pastinacasaliva), violets (Viola odorata), melon (Cucumis melo L.), zucchini(Cucurbita pepo L.), and pumpkin (Cucurbita pepo L). However, it is tobe understood that other plants may also be considered a plant ofinterest providing that they are not susceptible to the effects of P.tabacinum.

[0036] By “target plant” it is meant one or more plants that aresusceptible to the effects of the biocontrol agent and exhibit reducedgrowth, development or death when exposed to the biocontrol agent.Target plants are typically weed species, for example but not limited toherbicide-resistant and herbicide susceptible cleavers (Galium aparineL. and Galium spurium L.).

[0037]Plectosporium. tabacinum has been suggested as a potentialbioherbicide against arrowhead (Sagittaria trifolia L.), a weeddifficult to control in rice fields in Korea (Chung et al., 1998), andhydrilla (Hydrilla veilicillata (L.F.) Royle), an invasive aquatic weedin the Southeastern United States (Smither-Kopperl, 1999). Differencesin the growth characteristics and host specificity between the P.tabacinum strain of the present invention and these other isolatessuggest that the P. tabacinum fungal strain of the present invention isdifferent from the other isolates. However, the biocontrol agent of thepresent invention has not been suggested for use in non-aquaticenvironments, or with plants that are not exposed to periodicsubmersion. Therefore, in an aspect of an embodiment of the presentinvention, which is not meant to be considered limiting in any manner,there is provided the use of P. tabacinum, preferably P. tabacinumstrain (van Beyma) CL98-103, or more preferably, P. tabacinum depositedat the ATCC as PTA-3463 for the control of weeds under more aridconditions, that do not involve continuous, or prolonged water exposureregimes.

[0038] Referring now to FIG. 1, the fungal biocontrol agent as describedherein, grown on potato dextrose agar exhibits colonies that are moist,translucent white to pink in colour, radially furrowed with concentricrings and with a felty appearance. Further characteristics of fungalbiocontrol agent are described in Example 1.

[0039] Seedlings at the cotyledon or 1-whorl stages are at the mostsuitable stage for chemical application (Malik and Vanden Born, 1988,Phytopathol. 83:1229-1234). Referring now to FIG. 2, there is shown theeffect of P. tabacinum , in this case strain CL98-103, on false cleaversseedlings at the cotyledon stage (FIG. 2A) and one whorl stage (FIG.2B). The plants shown on the left are treated with a composition thatlacks P. tabacinum CL98-103 whereas plants on the right are treated withP. tabacinum CL98-103. The results indicate that fungal biocontrol agentP. tabacinum may be employed for the control of false cleavers. Further,as indicated in Table 1 of Example 2, P. tabacinum caused large necroticlesions on leaves and stems of herbicide-resistant andherbicide-susceptible false cleavers seedlings but was not pathogenic toherbicide-resistant or herbicide susceptible canola suggesting that thefungal strain of the present invention may be employed to control falsecleavers in canola.

[0040] Referring now to FIG. 3, the biocontrol agent of the presentinvention kills false cleavers seedlings when applied to seedlings at aconcentration of about 10⁷ spores per mL and with a dew period of about16 to 24 hours (FIG. 3A). However, other application doses incombination with other dew periods were capable of reducing the dryweight of false cleavers plants (FIG. 3B) suggesting that P. tabacinummay permit a plant of interest, for example, but not limited to canolato out compete false cleavers weeds in a field. Collectively, theseresults suggest that P. tabacinum may be employed as a biocontrol agentfor false cleavers weeds.

[0041] Referring now to FIG. 4, there is shown the effect of inoculumconcentration of P. tabacinum , for example P. tabacinum CL98-103, andcleaver leaf stage on the mortality and percent dry weight reduction offalse cleavers plants. As suggested by the results of FIG. 4A onehundred percent mortality of false cleavers is observed with an inoculumconcentration of 10 ⁷ conidia per mL and the cleavers growth stage isbetween about the cotyledon stage and the 1-whorl stage. However, otherinoculum doses also caused significant mortality of false cleaversplants. Further, as shown by the results depicted in FIG. 4B, variousinoculum concentrations when applied to false cleavers at growth stagesbetween about the cotyledon and 5 leaf stage exhibited a dry weightreduction of false cleavers suggesting that P. tabacinum CL98-103 maypermit a plant of interest to out-compete false cleavers weeds in afield. Preferably, the biocontrol agent of the present invention isapplied to false cleavers when the weeds are at a growth stage of aboutthe 1-whorl stage or earlier. Further, it is preferable that thebiocontrol agent of the present invention be applied at a concentrationof about 1×10⁷ conidia per mL or greater. However, increasing theinoculum concentration may be used in the biocontrol of older falsecleavers seedlings and such an application is fully contemplated by thepresent invention.

[0042] Therefore, the present invention provides a method for thebiocontrol of one or more weeds, for example, but not limited to falsecleavers, using the fungus P. tabacinum . Preferably the fungus is a P.tabacinum strain (van Beyma) CL98-103. More preferably, the fungus is P.tabacinum deposited at the ATCC as PTA-3463. Furthermore, the use of thebiocontrol agent as described herein may be supplemented with one ormore chemical herbicides as required.

[0043] Efficacy and host specificity are the two major criteria in theselection of suitable plant pathogens as weed biocontrol agents. Asdescribed in more detail in the examples, the fungus P. tabacinum causedmortality in false cleavers and cleavers seedlings. Therefore, P.tabacinum acts as a biocontrol agent and has the potential to controlcleavers. Moreover, P. tabacinum also exhibited similar effects onherbicide-resistant, and herbicide-susceptible cleavers (Galium aparineL. and G. spurium L).

[0044]P. tabacinum was non-pathogenic to canola, regardless of speciesand novel traits (herbicide tolerant vs conventional cultivars). Thus,P. tabacinum can be safely used in canola for false cleavers control.Further, P. tabacinum is non-pathogenic towards other commerciallyimportant plants, for example, but not limited to, wheat (Triticumaestivum L.), barley (Hordeum vulgare L.), oats (Avena sativa L.),alfalfa (Medicago sativa L.), pea, and lentil (Lens culinaris Medic.). Aminor response was observed in response to application of P. tabacinumto flax, safflower, melon, zucchini, and tomato when these plants weresubjected to a 24 h dew period treatment. However, the lesions coveredless than about 3% of the infected leaves and did not expand during aweek-long observation period. In total, host specificity tests on 35plant species in 26 genera and 12 families demonstrated that P.tabacinum is safe to use and that P. tabacinum is an effectivebiocontrol agent for cleavers (see Table 4, Example 2).

[0045] Pathogen inoculum concentration, plant growth stage, dew periodand temperature have been recognized as important pathogen, plant, andenvironment components that contribute to the efficacy of a bioherbicide(TeBeest, 1991, Ecology and epidemiology of fungal plant pathogensstudied as biological control agents of weeds. pp. 97-114 in TeBeest,D.O. (ed) Microbial Control of Weeds. Chapman & Hall, New York; Yang andTeBeest, 1993, Phytopathol. 83:891-893). Referring now to FIG. 5, thereis shown the effect of dew period duration on the mortality and dryweight reduction of false cleavers after spraying the weeds with about1×10⁷ spores per mL of P. tabacinum , in this example strain CL98-103.One hundred percent mortality of false cleavers seedlings was observedwith a dew period duration of about 16 hours or longer, but shorter dewperiods also promoted significant mortality of false cleavers (FIG. 5A).Further, dew periods of about 12 hours or longer promoted a dry weightreduction of false cleavers in the range of about 80% or greater, whiledew periods less than about 12 hours also resulted in substantial dryweight reduction of false cleavers.

[0046] The minimum dew period to achieve about 100% mortality of falsecleavers seedlings is dependent on temperature (Table 2, Example 2). Ata dew period temperature of about 22° C., the minimum dew period toachieve about 100% mortality of false cleavers was about 16 h (FIG. 5).This dew period requirement is similar to those reported for otherbioherbicides including Colletotrichum gloeosporioides fsp. malvae(TeBeest et al., 1978, Phytopathol. 68:389-393; Makowski, 1993,Phytopathol. 83: 1229-1234). Dew period requirements of 16 h or longermay limit the practical use of fungi as biological control agents forweeds. However, the addition of a surfactant, for example, but notlimited to Silwet L-77 in combination with the biocontrol agent of thepresent invention reduces the dew period for mortality of cleavers. Forexample, but not wishing to be limiting, the addition of about 0.05% toabout 0.1% Silwet L-77 decreased the minimum dew period for 100%mortality of G. spurium to 12 h. Eight-hour dew also caused more thanabout 70% mortality and greater than about 80% dry weight reduction.

[0047] Repetitive dew periods are known to shorten the optimal dewperiod required for mortality of weeds such as Senna obtusifolia byAlternaria cassiae and Echinochloa species by Exserohilum monoceras.Similar results are obtained with the biocontrol agent of the presentinvention. As described herein (Table 3, Example 2), multiple,repetitive dew periods, that more closely simulate field conditions thana single long dew period, exhibited enhanced control of false cleaversseedlings.

[0048] Therefore, P. tabacinum may introduce one or more novel modes ofaction to mitigate herbicide resistance development in cleavers and canbe considered as a component for herbicide resistant cleaversmanagement.

[0049] Radial mycelial growth, conidium production, and conidialgermination of P. tabacinum, for example but not limited to P. tabacinumCL98-103, responded differently to changes in nutritional andenvironmental conditions. Referring now to FIG. 6, there is shown theeffect of temperature on the growth and spore production of P. tabacinumCL98-103 on different agar media. The results suggest that a variety ofagar media, for example but not limited to potato dextrose agar,Czapek-Dox agar, lima bean agar, and V-8 juice agar may be employed forthe growth and spore production of P. tabacinum CL98-103. Other agartypes which may be used for growth and spore production of P. tabacinumCL98-103 include, but are not limited to oatmeal agar, tryptic soy agar,dextrose tryptone agar, Cooke rose bengal agar, prune agar, malt extractagar, synthetic nutrient poor agar, Sabouraud dextrose agar, water agarand cornmeal agar (Table 5, example 3). A preferred temperature rangefor mycelial growth was between about 20° C. and about 30° C., buttemperatures outside this range are also acceptable for growth of P.tabacinum CL98-103 (FIG. 6). An effective temperature for sporulationwas about 20° C. or about 30° C. depending on upon the nutrient medium.For example, but not wishing to be limiting, sporulation of P. tabacinumCL98-103 on, Czapek-Dox agar, lima bean agar, and V-8 juice agar washigh at a temperature of about 20° C., while sporulation on potatodextrose agar was high at a temperature of about 30° C.

[0050] Referring now to FIG. 7, there is shown the effect of medium pHon spore production of P. tabacinum , for example but not limited to P.tabacinum CL98-103, grown on potato dextrose agar (FIG. 7A) and indifferent liquid culture media (FIG. 7B). The results indicate that avariety of liquid culture media may be employed for the growth and sporeproduction of P. tabacinum , and that media of different pHs may beemployed for growth and spore production. As shown in FIG. 7B, sporeproduction observed for media with a pH in the range of about 6 to about8, was generally higher than spore production observed for the samemedia at lower pH values. The present invention contemplates growing P.tabacinum on any suitable solid medium or in any suitable liquid culturemedium. Further, the present invention contemplates spore production ofP. tabacinum on any suitable solid medium or in any suitable liquidculture medium. Also, the present invention contemplates formulations ofP. tabacinum , spores of P. tabacinum , or a combination thereof on anysuitable solid medium or in any suitable liquid culture medium.Preferably the P. tabacinum is strain (van Beyma) CL98-103.

[0051] Referring now to FIG. 8, there is shown the effect of thecarbon:nitrogen (C:N) ratio of the medium on spore production of P.tabacinum for medium comprising 8.4 (FIG. 8A), 21 (FIG. 8B) and 33.6gL⁻¹ sucrose (FIG. 8C). The results shown in FIG. 8 indicate thatdifferent media, comprising a range of sugars, for example, but notlimited to sucrose and exhibiting a range of C:N ratios may be employedfor the growth and spore production of P. tabacinum. Without wishing tobe limiting nitrogen sources may include, but are not limited topotassium nitrate, corn gluten meal, corn steep liquor, glutamic acid,asparagines, casamino acids, yeast extract, sodium nitrate, casein,urea, malt extract, ammonium sulfate, bovine serum albumin, cottonseedoil, or a combination thereof Carbon sources may comprise, but are notlimited to sucrose, galactose, corn starch, cellulose, glucose,fructose, citric acid or a combination thereof

[0052] As described in Example 3, P. tabacinum produces large quantitiesof spores in liquid fermentation medium within 3 d when the initialspore concentration is about 5×10⁴ spore/ml. Thus, spore production isnot a limiting factor for the development of this fungus as abioherbicide for control of cleavers.

[0053] Thus, in an aspect of an embodiment of the present invention,there is provided a method of controlling cleavers (Galium aparine L.and G. spurium L) that comprises applying an effective amount ofbiocontrol agent P. tabacinum to the cleavers. Preferably the P.tabacinum is strain (van Beyma) CL98-103. The cleavers may compriseherbicide-resistant cleavers, herbicide-susceptible cleavers or acombination thereof Further, the present invention contemplatespreventative control of cleavers, for example, but not limited to,applying an effective amount of biocontrol agent P. tabacinum ,preferably P. tabacinum strain (van Beyma) CL98-103, to preventemergence of cleavers. Preferably, the effective amount of P. tabacinumstrain CL98-103 is in the range of about 1×10⁵ to about 1×10⁸ spores permL and the cleavers are sprayed until runoff. However, sporeconcentrations outside this range may be employed if desired.

[0054] The biocontrol agent of the present invention may be formulatedin an acceptable carrier, for example, but not limited to a solid orliquid growth medium, preservation medium or the like. Acceptablecarriers for spores may comprise, but are not limited to clay, alginate,diatomaceous earth or liquids containing suitable adjuvants, forexample, but not limited to potato dextrose agar (PDA), gelatin, aminoacids, sugars, surfactants and other solutes. Any carrier that permitsthe biocontrol agent to remain viable and pathogenic may be employed inthe method of the present invention.

[0055] The above description is not intended to limit the claimedinvention in any manner, furthermore, the discussed combination offeatures might not be absolutely necessary for the inventive solution.

[0056] The present invention will be further illustrated in thefollowing examples. However it is to be understood that these examplesare for illustrative purposes only, and should not be used to limit thescope of the present invention in any manner.

EXAMPLES Example 1 Isolation, Identification and Characterization ofBio-Control Agent CL98-103.

[0057] Diseased cleavers were collected in the districts of Vermilion,Vegreville, Lamont, Edmonton, and Peace River, Alberta, Canada. Diseasedleaves, stems, flowers and seeds were air dried in a paper press, cut toappropriate size, and stored at 4° C. in envelopes. Tissue pieces withlesions were surface sterilized with 0.5% sodium hypochlorite solutionand incubated on fresh potato dextrose agar (PDA; Difco, Detroit,Mich.). Fungi that grew from the lesions were isolated and Koch'spostulates were performed for most samples shortly after each collectiontrip. Single conidial isolates of the recovered fungi were maintained incryovials, each containing 2 ml of individual fungal isolate in 15%glycerol, and stored at −80° C. as stock cultures. From those collectedplant materials, 138 fungal pathogens were isolated. A fungal isolateCL98-103 was selected after extensive screening on virulence to cleaversand safety to nine major crops (canola, wheat, barley, oats, flax,safflower, alfalfa, field pea, and lentil).

[0058] Inoculum Production

[0059] A cryovial of stock culture was warmed to room temperature in awater bath at 36° C., aseptically placing about 200 μl of suspension onthe surface of a potato dextrose agar (PDA) Petri plate and spreadingconidia with a sterile glass rod. Petri plates were sealed and incubatedat 22° C. with a 12 h photoperiod for 5-8 days. Single-conidium colonieswere then made from the actively growing cultures and incubated underthe same conditions. Conidia from the single-spore cultures wereaseptically spread onto PDA and incubated as described above to increaseinoculum. Conidia were harvested 15 days after incubation by floodingthe plates with 10 ml of distilled water and scraping the surface of thecolonies with a glass slide. The resulting suspensions were filteredthrough a layer of cheesecloth and conidial concentrations weredetermined with a hemacytometer.

[0060] Conidia were also produced in Richard's solution (RS; 50 gsucrose, 10 g KNO₃, 5 g KH₂PO₄, 2.4 g MgSO₄, 0.02 g FeCl₃, 1 L distilledwater). A bulk of RS was prepared, adjusted pH to 7.0 using 1N NaOH orHCl, and distributed 200 ml into 500-ml Erlenmeyer flasks. Flaskscontaining the RS were then autoclaved for 15 min (100 kPa and 121° C.).After cooling, each flask was inoculated with ‘seed’ inoculum of P.tabacinum CL98-103. Inoculated flasks were incubated on an orbit shakerat about 150 rpm under ambient laboratory conditions (24±3° C.). After 3d incubation, conidia were harvested by grinding the content of eachflask using an electric hand blender (Braun Multipractic MR 20,Lunnfield, Mass., USA) and centrifuging 10 min to form a pellet (SorvallRC-5B refrigerated superspeed centrifuge). The supernatant was decantedoff and the conidial pellet was resuspended in an appropriate amount of1% gelatin to achieve the desired inoculum concentration as determinedwith the aid of a haemocytometer.

[0061] Mycelial and spore characteristics of biocontrol agent CL98-103were described through direct unaided inspection and microscopicobservation. Both a light microscope (Nikon Inc., Melville, N.Y., USA)and scanning electron microscopy (SEM) (JEOL 6301F field emission SEM)were used. Colonies of CL98-103 grown on potato dextrose agar weremoist, translucent white to pink in color, radially furrowed withconcentric rings, and exhibited a felty appearance (aerial hyphaeloosely aggregated into strands) (FIGS. 1A, 1B). CL98-103 is furthercharacterized by simple or branched conidiophores and apical or lateralphialides that sometimes proliferate percurrently or have more than oneconidiogenous locus with a cylindrical collarette and sinuous apex (FIG.1C). Conidia are slightly asymmetric and thus appear slightly curved,hyaline, and 0 to 1 septate (FIGS. 1D, 1E). Germination can be bilateralor monolateral (FIGS. 1F, 1G, 1H).

[0062] The fungal isolate CL98-103 was identified as Plectosporiumtabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, an anamorph ofPlectoshphaerella cucumerina (Lindf) W. Gams, at Centraalbureau voorSchimmelcultures, AG Baam, The Netherlands. The strain was deposited inthe ATCC as PTA-3463.

Example 2 Pathogenicity of P. tabacinum CL98-103.

[0063] Effectiveness of P. Tabacinum CL98-103 to Control False Cleavers

[0064] Both Argentine canola and Polish canola with or without noveltraits were selected for the pathogenicity test of P. tabacinumCL98-103. Three herbicide-tolerant cultivars, Invigor 2153 (LibertyLink), Quest (Roundup Ready), and 45A71 (Pursuit Smart), and twoconventional cultivars Quantum and Impulse were selected asrepresentatives of Argentine canola, while Hysin 111 and Reward wereselected as representatives of Polish canola. For false cleavers, bothherbicide-resistant and herbicide-susceptible biotypes were included inthe pathogenicity test. False cleavers and canola cultivars were grownin greenhouse pots with 24/20±5° C. day/night temperature, a 16 hphotoperiod, an average light intensity of 300 μEm⁻²s⁻¹, and an averagerelative humidity of 45-50%. False cleavers seedlings at the one-whorlstage and canola seedlings at the one- or two-leaf stage were sprayeduntil run-off with 10⁶ to 10⁷ conidia per ml in 1% gelatin solution(Difco, Setroit, Mich., USA), using an H-set airbrush (Paasche AirbrushCompany, Harwood Heights, Ill.) at a pressure of 100 kPa. Control plantswere sprayed with 1% gelatin only. About 30 min after spraying, potswere placed in a dark dew chamber at 100% relative humidity at 22° C.for 24 h. Subsequently, pots were returned to the greenhouse for theremainder of the experiment. The disease reactions ofherbicide-resistant and herbicide-susceptible false cleavers and canolato P. tabacinum CL98-103 were evaluated seven days after inoculation. Onthe basis of lesion type and size, plant response was rated at fourlevels: 0=lesions absent; 1=small, unexpanded lesions; 2=slightly tomoderately expanded lesions; and 3=large lesions or dead plants. Dryweight was obtained by cutting aerial parts of soil level, drying inpaper bags for 48 h at 70° C., and weighing.

[0065] Results showed that P. tabacinum CL98-103 caused large, necroticlesions on leaves and stems of both herbicide-resistant andherbicide-susceptible false cleavers seedlings. The disease rating was 3on both herbicide-resistant and herbicide-susceptible false cleavers(Table 1). P. tabacinum CL98-103, however, was not pathogenic to fivecultivars of Argentine canola and two cultivars of Polish canola (Table1). Without wishing to be bound by theory, the results suggest thatcanola is not a host of P. tabacinum , and that P. tabacinum CL98-103may be safely used for the control of false cleavers in canola and othercrops. TABLE 1 Pathogenicity of Plectosporium tabacinum CL98-103 onherbicide-resistant and herbicide-susceptible false cleavers and variouscultivars of canola* Plants Host Galium spurium (False cleavers)cleavers) Herbicide resistant type 3 Herbicide susceptible type 3Brassica napus (Argentine Canola) cv. Invigor 2153 (Liberty Link) 0 cv.Quest (Roundup Ready) 0 cv. 45A71 (Pursuit Smart) 0 cv. Quantum(Conventional) 0 cv. Impulse (Conventional) 0 Brassica rapa (Canola)Hysin 111 0 Reward 0

[0066] The efficacy of P. tabacinum CL98-103 as a biocontrol agenttowards false cleavers was determined under single or combined factorssuch as inoculum concentration, weed growth stage, dew period,temperature, and surfactant.

[0067] Effect of Conidial Concentration and Dew Period Duration on theAbility of P. tabacinum CL98-103 to Control False Cleavers

[0068] The efficacy of P. tabacinum on both herbicide-resistant andherbicide-susceptible false cleavers was assessed under differentconidial concentrations and dew period durations. Bothherbicide-resistant and herbicide-susceptible false cleavers seedlingswere produced in greenhouse pots, sprayed with a conidial concentrationof 5×10⁶, 1×10⁷, 5×10⁷, or 1×10⁸ conidia per ml in 1% gelatin solution,placed in the dark dew chamber with 100% relative humidity at 22° C. for8, 12, 16, or 20 h, and subsequently returned to the greenhouse. Sevendays after spraying, mortality of plants and dry weight of abovegroundbiomass per pot were assessed. The P. tabacinum CL98-103 strain of thisinvention killed false cleavers seedlings when applied at aconcentration of 107 conidia per ml or greater with a 16-20 h dew period(FIG. 3). P. tabacinum CL98-103 therefore has potential for use as abioherbicide for control of false cleavers. Another significantcharacteristic of P. tabacinum CL98-103 is its ability to killherbicide-resistant false cleavers under the same conditions. TheEfficacy of P. tabacinum CL98-103 to herbicide-resistant false cleaversseedlings was identical to herbicide-susceptible false cleaversseedlings in terms of plant mortality and percent dry weight reduction.Thus, the use of P. tabacinum CL98-103 may provide an option to manageherbicide-resistant false cleavers.

[0069] Effect of Conidial Concentration and Weed Growth Stage on Abilityof P. tabacinum CL98-103 to Control False Cleavers

[0070] The efficacy of P. tabacinum CL98-103 on false cleavers wasassessed under different conidial concentrations and weed growth stages.Seedlings at the cotyledon, and 1-, 2-, 3-, 4-, or 5-whorl growth stageof false cleavers were inoculated with a conidial suspension atconcentrations of 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵ or 0 conidia/ml. Afterinoculation, pots with inoculated seedlings were placed in a dark dewchamber for 24 h and then returned to the greenhouse. One hundredpercent mortality of false cleavers seedlings was observed with theplant growth stage at the cotyledon- to 1-whorl stage and with aninoculum concentration of 107 conidia/ml or greater (FIG. 4). Theminimum inoculum concentration required to kill false cleavers seedlingswas 1×10⁷ conidia/ml. The 1-whorl growth stage or younger of falsecleavers was the most susceptible. Increasing inoculum concentrationincreased weed control efficacy on older false cleavers seedlings.

[0071] Effect of Dew Period Duration on Ability of P. tabacinum CL98-103to Control False Cleavers

[0072] The efficacy of P. tabacinum CL98-103 on false cleavers wasassessed under different dew period durations. False cleavers seedlingswere produced in greenhouse pots, sprayed with a conidial concentrationof 1×10⁷ spores per ml in 1% gelatin solution, placed in the dark dewchamber with 100% relative humidity at 22° C. for 0, 4, 8, 12, 16, 20,or 24 h. After the dew period treatment, pots were returned to thegreenhouse. Seven days after spraying, mortality of plants and dryweight of aboveground biomass per pot were assessed. When adequate dewwas provided, 100% mortality and dry weight reduction occurred (FIG. 5).The minimum dew period to achieve 100% mortality is 16 h without anyformulation.

[0073] Effect of Dew Point Duration and Surfactant Concentration onAbility of P. tabacinum CL98-103 to Control False Cleavers

[0074] The efficacy of P. tabacinum CL98-103 on false cleavers wasassessed under different dew period durations and various concentrationsof surfactant Silwet L-77. Since the minimum dew period to achieve 100%mortality is 16 h without any formulation, dew period may be a limitingfactor for P. tabacinum CL98-103 to control false cleavers. Therefore,it is important to know whether a formulation with surfactant can reducethe dew period requirement for P. tabacinum CL98-103 to control falsecleavers. False cleavers seedlings were produced in greenhouse pots,sprayed with a conidial concentration of 1×107 conidia per ml in 1%gelatin solution plus 0, 0.05%, or 0.1% Silwet L-77, placed in the darkdew chamber with 100% relative humidity at 22° C. for 0, 4, 8, 12, 16,20, or 24 h. After the dew period treatment, pots were returned to thegreenhouse. Seven days after spray, mortality of plants and dry weightof aboveground biomass per pot were assessed. Addition of 0.05-0.1%Silwet L-77 significantly reduced the minimum dew period for 100%mortality of false cleavers. An 8-h dew caused more than 70% mortalityand over 80% dry weight reduction. When 12-h dew was provided, 100%mortality and dry weight reduction occurred. These results suggest thata formulation comprising the biocontrol agent of the present inventionand Silwet L-77 may overcome the dew point limiting factor.

[0075] Effect of Dew Period Temperature and Duration on Mortality andReduction in Dry Weight of False Cleavers Inoculated with Plectosporiumtabacinum CL98-103

[0076] The efficacy of P. tabacinum CL98-103 on false cleavers wasassessed under different dew period temperatures and durations. Falsecleavers seedlings at the 1-whorl stage were inoculated with conidialsuspensions at a concentration of 1×10⁷ conidia/ml. After inoculation,pots with inoculated seedlings were placed in the dew chamber at 100%humidity and temperatures of 15, 20, or 22° C. for 16 or 24 h and thenreturned to the greenhouse. Dew period temperature significantlyaffected the mortality and dry weight reduction of false cleaversseedlings caused by P. tabacinum CL98-103 (Table 2). When dew periodtemperature was 22° C., 100% mortality and dry weight reduction of falsecleavers seedlings were observed with both 16 h and 24 h dew perioddurations. When dew period temperature was 20° C., a similar result wasobserved, but mortality and dry weight reduction of false cleaversseedlings with 24 h dew was greater than those with 16 h dew. When dewperiod temperature was 15° C., mortality and dry weight reduction offalse cleavers seedlings were significantly less than those observed atdew period temperature of 20 or 22° C. Dew period temperature of 15° C.did not cause any mortality with 16 h dew, but resulted more than 86%mortality and dry weight reduction of false cleavers seedlings. Aneffective dew period temperature for disease development was above 15°C. TABLE 2 Effect of dew period temperature and duration on mortalityand reduction in dry weight of false cleavers inoculated withPlectosporium tabacinum* Dew period duration 16 h 24 h Percent PercentDew period dry weight dry weight temperature Mortality reductionMortality reduction (° C.) (%) (%) (%) (%) 15 0 21.7 86.7 87 20 90.3 9893 99 22 100 100 100 100

[0077] The Effect of Multiple Repetitive Dew Periods on the Ability ofP. tabacinum CL98-103 to Control False Cleavers

[0078] The efficacy of P. tabacinum CL98-103 on false cleavers wasassessed under multiple, repetitive dew periods. After inoculation, potswith inoculated seedlings were provided one of the following dark dewperiods: (1) 0-h dew, (2) 8-h dew/16-h dry period on 1, 2, 3 or 4consecutive days, (3) 12-h dew/12-h dry period on 1 or 2 consecutivedays, (4) 16-h dew/8-h dry on 1 or 2 consecutive days; or (5) a single24-h dew period. After treatment, pots were returned to the greenhouse.When provided with a continuous dew of 16-h or more, P. tabacinumCL98-103 caused 90-100% mortality and 92-100% dry weight reduction offalse cleavers seedlings (Table 3). Repetitive 8-h dew on 1, 2, 3, or 4consecutive days showed a general increase in dry weight reduction ofcleavers. Two 12-h dew on consecutive days increased mortality of falsecleavers seedlings, causing 83% mortality compared with 53% mortalitywith single 12-h dew. Similarly, two 12-h dew on consecutive daysincreased percent dry weight reduction (90%) as compared to single 12-hdew. The results suggest that disease development increases wheninoculated seedlings are subjected to multiple, repetitive dew periodsand that longer dew periods are more effective than shorter dew periods.TABLE 3 Effect of repetitive dew periods on mortality and reduction indry weight of false cleavers inoculated with Plectosporium tabacinumCL98-103* Dew period Dry weight (h/day) Mortality (%) reduction (%)  0 0c** 10 c  8 0 c 15.6 c 8; 8 0 c 28 bc 8; 8; 8 0 c 40.4 bc 8; 8; 8; 8 0 c45 b 12 53 b 77.4 a 12; 12 83 a 90.1 a 16 90 a 92.4 a 24 100 a 100 a

[0079] Host Range of P. tabacinum CL98-103.

[0080] Using the modified centrifugal phylogenetic and varietal strategy(Wapshere, 1974), 35 plant species in 26 genera and 12 families wereselected for the host range trial (Table 4). Test plants were grown fromseed or propagated vegetatively depending on the species being testedand availability of material. For each species, two sets of test plants(each set containing 15 plants) were prepared. One set was inoculatedwith P. tabacinum CL98-103 and the other set served as uninoculatedcontrols. For the inoculated treatment, seedlings of selected plantspecies at the 2- to 3-leaf stage were sprayed to run-off with 1×10⁷conidia per ml in 1% gelatin solution. For uninoculated control,seedlings of selected plant species at the 2- to 3-leaf stage weresprayed with 1% gelatin. About 30 min after spraying, pots were placedin the dark dew chamber with 100% relative humidity at 22° C. for 24 h.Subsequently, pots were returned to the greenhouse for the remainder ofthe experiment. Disease severity was visually assessed daily until the₁₄ ^(th) day after inoculation by using the 0-11 rating scale byHorsfall and Barrett (1945).

[0081] All seedlings of false cleavers serving as positive controls werekilled when subjected to a 24 h dew period. Cleavers (Galium aparineL.), another weed species in the Galium weed complex in western Canada,was also killed with 24 h dew. The findings in this study demonstratethat P. tabacinum CL98-103 may be used for the control of two weedyGalium species. Results also showed that other species in the genusGalium are immune to P. tabacinum . Hosts of P. tabacinum CL98-103 inthe Rubiaceae family were restricted to the Section Kolgyda (falsecleavers and cleavers) in the Genus Galium, the tribe Rubieae, and thesubfamily Rubiodieae. The use of this strain of P. tabacinum shouldtherefore be safe to non-weedy native Galium species and other plantspecies in this family.

[0082]P. tabacinum CL98-103 caused slight symptoms with limitedexpansion of lesions on sunflower (Helianthus annuus L.) with the 24 hdew period treatment. Lesion expansion covered less than 10% ofinoculated leaves 2 weeks after inoculation and no stem lesions wereobserved. Lesions did not spread to emerging uninoculated leaves.Similarly, P. tabacinum CL98-103 caused small (<0.5 mm) brown flecks oninoculated leaves of flax (Linum usitatissimum L.), safflower (Carthamustinctorius L.), melon (Cucumis melo L.), zucchini (Cucubita pepo L.),and tomato (Lycopersicon esculentum Mill.) with a 24 h dew periodtreatment. However, these small flecks did not expand during the 2-weekobservation period and lesions covered less than 3% of infected leaves.

[0083] All other plant species tested were immune to P. tabacinumCL98-103. Further, P. tabacinum has never been reported to cause cropdiseases in Canada. The detection of natural infection of P. tabacinumCL98-103 on cleavers but not on any crops in western Canada suggeststhat this fungus can be used as a bioherbicide to control cleavers.TABLE 4 Test plant species used for host-specificity screening ofPlectosporium tabacinum CL98-103 against false cleavers (Galium spurium)based on the modified centrifugal phylogenetic and varietal strategyRubiaceae  1. Galium spurium L. (False cleavers)  2. G. aparine L.(Cleavers)  3. G. boreale L. (Northern bedstraw)  4. G. triflorum Michx.(Fragrant bedstraw)  5. G. trifidum L. (Three petal bedstraw)  6. G.mollugo L. (Common hedge bedstraw)  7. G. trifidum L. (Three petalbedstraw)  8. Asperula arvensis L. (Blue woodruff)  9. Sherardiaarvensis L. (Blue field madder) 10. Houstonia longifolia L. (Longleafsummer blue) 11. Cephalanthus occidentalis L. (Button bush) Brassicaceae12. Brassica napus L. (Argentine Canola) cv. Invigor 2153 (Liberty Link)cv. Quest (Roundup Ready) cv. 45A71 (Pursuit Smart) cv. Quantum(Conventional) cv. Impulse (Conventional) 13. Brassica rapa L. (PolishCanola) cv. Hysyn 111 cv. Reward 14. Brassica oleracea L. var. botrytisL. (Cauliflower, cv. Snowball A) Poaceae 15. Triticum aestivum L.(Wheat, cv. Katepwa) 16. Hordeum vulgare L. (Barley, cv. Bridge) 17.Avena sativa L. (Oats, cv. Unknown) Fabaceae 18. Pisum sativum L. (Pea,cv. Radley) 19. Medicago sativa L. (Alfalfa, cv. Algonquin) 20. Lensculinaris Medic. (Lentil, cv. Laird) Linaceae 21. Linum usitatissimum L.(Flax, cv. Norlin) Asteraceae 22. Helianthus annuus L. (Sunflower cv.S6140) 23. Carthamus tinctorius L. (Safflower cv. unknown) 24. Taraxiumofficinale Weber in Wiggers (Dandelion) Solanaceae 25. Solanum tuberosumL. (Potato cv. Normondy cv. Russet burbank) 26. Lycopersicon esculentumL. (Tomato cv. Bush Beef Steak) 27. Nicotiana tabacum L. (Tobacco cv.Turkish) Balsaminaceae 28. Impatiens balsami L. (Balsam) Apiaceae 29.Apium graveolens L. (Celery cv. Utah Tall Green) 30. Pastinaca sativa(Parsnip cv. All American) Violaceae 31. Viola odorata (English violet)Cucurbitaceae 32. Cucumis melo L. (Melon cv. Hales Best) 33. Cucurbitapepo L. (Zucchini cv. Summer Squash) 34. Cucurbita pepo L. (Pumpkin cv.Big Mac) Asclepiadaceae 35. Asclepias sp. (Milkweed)

Example 3 Establishment of Culture Conditions for Enhanced Growth andSpore Production of P. tabacinum CL98-103

[0084] Culture conditions were varied to examine the growth, sporegermination, and sporulation requirements of P. tabacinum CL98-103 onstandard agar media. Fourteen different standard agar media over a rangeof light regimes, pH, and temperatures were tested. Culture media testedwere water agar (WA), Potato dextrose agar (PDA), Cooke rose bengal agar(CRBA), dextrose tryptone agar (DTA), Czapek Dox agar (CDA), tryptic soyagar (TSA), malt extract agar (MEA), sabouroud dextrose agar (SDA),oatmeal agar (OMA), prune agar (PA), lima bean agar (LBA), cornmeal agar(CA), V-8 juice agar (VA), and synthetic nutrient poor agar (SNA).Results demonstrated that PDA is a good medium for growth andsporulation of P. tabacinum CL98-103 (Table 5). On standard agar media,growth and sporulation of P. tabacinum was not appreciably influenced bylight regime. An effective temperature for mycelial growth was betweenabout 22 and about 25° C., but an effective temperature for sporulationwas either about 20 or about 30° C., depending upon the nutrient medium(FIG. 7). It appeared that between 15 and 20° C. changes in the sporegermination capabilities of this isolate occurred as over 90% of sporesgerminated when temperatures were about 20° C. or above, while less than10% of spores germinated when temperatures were about 15° C. or below.TABLE 5 Effect of culture medium and light regime on mycelial growth andsporulation of Plectosporium tabacinum CL98-103. Number of Colonyspores/plate diameter (mm) (× 10⁸) Medium Light Dark Light Dark Potatodextrose agar 63.3 54.2 6.942 6.458 V-8 juice agar 76   71.8 2.113 1.653Czapek-Dox agar 66.3 67.5 1.046 0.933 Oatmeal agar 62.7 61.8 0.818 0.941Lima bean agar 72.3 69.8 0.588 0.535 Tryptic soy agar 54.8 57.4 0.5610.208 Dextrose Tryptone agar 54.2 55.7 0.099 0.159 Cooke rose bengalagar 22.8 40.2 0.028 0.092 Prune agar 67.7 63.5 0.021 0.014 Malt extractagar 57.3 56.2 0.008 0.008 Synthetic nutrient-poor agar 74.3 72.8 0.0060.007 Sabouraud dextrose agar 38.8 39   0.006 0.006 Water agar 64.5 62.70.005 0.003 Cornmeal agar 72.7 72.7 0.004 0.006

[0085] Submerged liquid culture spore production is the preferredtechnique for mass production of biocontrol agents because thetechnology is readily available and the scale-up process from theresearch phase to the development phase is relatively easy.

[0086] Conditions required for submerged liquid culture spore productionand resulting weed control efficacy of P. tabacinum CL98-103 wasassessed, including the effect of liquid culture medium and pH and theeffect of carbon-nitrogen composition such as carbon source, nitrogensource, carbon concentration, and carbon-to-nitrogen ratio.

[0087] Liquid culture media tested included: Tryptic Soy broth (TSB),Czapek-Dox broth (CDB), Yeast Extract broth (YEB), Nutrient Broth (NB),Potato Dextrose broth (PDB), Malt Extract broth (MEB), Richard'ssolution (RS), modified Richard's solution (MRS) (Daniel et al. 1973),Colletotrichum truncatum medium (CTM) (Jackson 1997), V-8 juice medium(VM), and Trichoderma medium (TM) (Tabachnik 1989). The pH of culturemedia was not adjusted. Results indicate that a variety of liquidculture media may be employed for spore production of P. tabacinumCL98-103 (Table 6). TABLE 6 Effect of liquid culture medium on sporeproduction and efficacy against false cleavers of Plectosporiumtabacinum CL98-103*. Spore production Efficacy** Medium (10⁷ spores/ml)(%) V-8 juice medium (VM) 2.62 84   Modified Richard's solution (MSR)2.22 60.5 Yeast extract broth (YEB) 1.43 79.4 Richard's solution (RS)1.06 97.1 Czapek-Dox broth (CDB) 0.49 62.1 Trichoderma medium (TM) 0.31100   Tryptic soy broth (TSB) 0.3  ND Potato dextrose broth (PDB) 0.16ND Nutrient broth (NB) 0.05 ND Colletotrichum truncatum medium (CTM)0.04 ND Malt extract broth (MEB) 0.02 ND

[0088] Liquid culture medium pH also profoundly affected submergedliquid culture spore production of P. tabacinum CL98-103 (FIG. 7). Forexample, when the pH of RS remained unadjusted at 3.9 as in the liquidculture medium study, only 1.06×10⁷ spores/ml were produced (Table 6),while spore production in RS adjusted to pH 7 prior to inoculation wasabout seven times higher (7.7×10⁷ spores/ml; FIG. 7). An effective pHfor spore production of P. tabacinum CL98-103 is 7 for RS medium.

[0089] Liquid culture medium carbon-nitrogen composition such as carbonsource, nitrogen source, carbon concentration, and carbon-to-nitrogenratio was also varied to evaluate their effects on spore production,spore characteristics, and weed control efficacy of P. tabacinumCL98-103. Fourteen different nitrogen sources were tested including corngluten meal (12.42 gL⁻¹), corn steep liquor (23.99 gL⁻¹), malt extract(25 gL⁻¹), casein (10.1 gL⁻¹), yeast extract (13.24 gL⁻¹), bovine serumalbumin (9.06 gL⁻¹), cottonseed oil (77.12 gL⁻¹), casamino acids (14.04gL⁻¹), sodium nitrate (8.51 gL⁻¹), aspargine (6.62 gL⁻¹), glutamic acid(14.74 gL⁻¹), urea (3 gL⁻¹), ammonium sulfate (6.62 gL⁻¹), and potassiumnitrate (10 gL⁻¹). Ten different carbon sources were tested includingglucose, fructose, galactose, glycerol, potassium acetate, acetic acid,cellulose, corn starch, citrate, and sucrose. Eight carbonconcentrations of the medium (i.e. sucrose) were tested including 4.2,8.4, 12.6, 16.8, 21.0, 25.2, 29.4, or 33.6 g/L. Six C:N ratios, i.e.40:1, 20:1, 15:1, 10:1, 7.5:1, and 5:1, were evaluated under threedifferent levels of carbon concentration, i.e. 8.4, 21.0, and 33.6 gL⁻¹.Results demonstrated that the carbon-nitrogen composition of the mediumincluding nitrogen source, carbon source, carbon concentrations, and C:Nratios influences spore production, spore size, spore nuclear number,and/or weed control efficacy of P. tabacinum CL98-103. It has also beenfound that the carbon-nitrogen composition of the medium that allowedthe production of the highest number of spores did not result in theproduction of spores with the highest efficacy against false cleaverswhen spores were applied to plants at the same concentration and underthe same experimental conditions. For production of a high number of P.tabacinum CL98-103 spores with good weed control efficacy, a goodnitrogen source and carbon source may be potassium nitrate (Table 7) andsucrose or cornstarch (Table 8), respectively. Sucrose as the carbonsource produced moderate spore numbers (2.45×10⁷ spores/ml), but sporesproduced in this carbon source caused 99.8% dry weight reduction offalse cleavers. Spore production using corn starch (3.68×10⁷ spores/ml)was close to the highest spore numbers, but spores produced with cornstarch caused a percent dry weight reduction of false cleavers (94.6%)similar to that of sucrose (99.8%). Since corn starch is anagricultural-based product and readily available at a low cost, it maybe a good carbon source for mass production of this potentialbioherbicide.

[0090] A medium comprising a high carbon concentration and a low carbonconcentration inhibited spore production of P. tabacinum CL98-103 (Table9). A carbon concentration of 12.6 g/L produced high spore numbers andspores produced at this carbon concentration caused the significant dryweight reduction of false cleavers. The C:N ratio affected sporeproduction but not weed control efficacy. A C:N ratio of 7.5:1 iseffective for spore production of P. tabacinum CL98-103 (FIG. 8). Basedon the results, an effective liquid medium for production of a highnumber of P. tabacinum CL98-103 spores with good weed control efficacymay be constructed using a C:N ratio of 7.5:1 with 12.6 g/L sucrose orcorn starch, and 10 g/L potassium nitrate. TABLE 7 Effect of nitrogensource on spore production, spore nuclear number, spore size, andefficacy against false cleavers of Plectosporium tabacinum CL98-103 in abasal salts medium based on Richard's solution* Spore production SporeSpore Nitrogen (10⁷ Nuclear length width Efficacy* sources spores/ml)number (μm) (μm) *(%) potassium 2.45 1.8 7.4 3.4a 100 nitrate (control)6.63 1.6 7.4 3.2 80.8 corn gluten meal corn steep 2.7 1.7 7.1 3 91.7liquor glutamic acid 1.72 1.9 7.4 3.3 52.9 asparagines 1.67 1.6 7 3 50.7casamino acids 1.54 1.8 7.6 3.1 93.5 yeast extract 1.54 1.8 7.5 3.4 100sodium nitrate 1.48 1.9 7.3 3.2 65.6 casein 0.83 ND ND ND ND urea 0.66ND ND ND ND malt extract 0.2 ND ND ND ND ammonium 0.13 ND ND ND NDsulfate bovine serum 0.12 ND ND ND ND albumin cottonseed oil 0 ND ND NDND

[0091] TABLE 8 Effect of carbon source on spore production, sporenuclear number, spore size, and efficacy against false cleavers ofPlectosporium tabacinum CL98-103 in a basal salts medium based onRichard's solution* Spore production Spore Spore Carbon (10⁷ Nuclearlength width Efficacy** sources spores/ml) number (μm) (μm) (%) Sucrose2.45 1.9 8.3 3.9 99.8 (control) galactose 3.81 1.7 7.6 4.4 62.4 cornstarch 3.68 1.8 6.3 3.4 94.6 cellulose 1.62 1.5 6.9 3   46 glucose 0.091.4 6.9 3.9 63.5 fructose 0.08 1.4 7.3 3.9 96 citric acid 0.06 1.7 8.13.7 98.9 glycerol 0.004 ND ND ND ND potassium 0.002 ND ND ND ND acetateacetic acid 0 ND ND ND ND

[0092] TABLE 9 Effect of carbon concentration on spore production, sporenuclear number, spore size, and efficacy against false cleavers ofPlectosporium tabacinum in a basal salts medium based on Richard'ssolution Carbon Spore concen- production tration (10⁷ Nuclear SporeSpore Efficacy** (g/L) spores/ml) number length (μm) width (μm) (%) 4.25.23 1.6 7.2 3 58.8 8.4 6.25 1.7 7.6 3.2 81.9 12.6 6.77 1.7 7.5 3.1 90.016.8 5.68 1.9 7.6 3.2 88.3 21 5.08 1.7 7.2 3.3 83.5 25.2 4.65 1.7 7.4 390.8 29.4 4.37 1.7 7 3.3 83.4 33.6 3.68 1.8 7 3.1 81.9

[0093] All citations are herein incorporated by reference.

[0094] The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

[0095] References

[0096] Each of the below references are hereby incorporated by referencefor any purpose. Chung, Y. R., S. J. Koo, H. T. Kim, and K. Y. Cho.1998. Potential of an indigenous fungus, Plectosporium tabacinum, as amycoherbicide for control of arrowhead (Sagittaria trifolia). Plant Dis.82: 657-660.

[0097] Hall, L. M, K. M. Stromme, G. P. Horsman, and M. D. Devine. 1998.Resistance to acetolactate synthase inhibitors and quinclorac in abiotype of false cleavers (Galium spurium). Weed Sci. 46:390-396.

[0098] Horsfall, J. G. and R. W. Barrett. 1945. An improved gradingsystem for measuring plant diseases. Phytopathol. 35:655.

[0099] Malik, N. and W. H. Vanden Born. 1987. Growth and development offalse cleavers (Galium spurium L.). Weed Sci. 35:490-495.

[0100] Malik, N. and W. H. Vanden Born. 1988. The biology of Canadianweeds. 86. Galium aparine L. and Galium spurium L. Can. J. Plant Sci.68: 481-499.

[0101] Smither-Kopperl, M. L., R. Charudattan, and R. D. Berger. 1999.Plectosporium tabacinum, a pathogen of the invasive aquatic weedHydrilla verticillata in Florida. Plant Dis. 83: 24-28.

[0102] Thomas, A. G., B. Frick, and L. M. Hall. 1998. Weed populationshifts in Alberta. Agriculture and Agri-Food Canada, Saskatoon, pp. 1.

We claim:
 1. A biocontrol agent comprising Plectosporium. tabacinumCL98-103.
 2. A composition comprising the biocontrol agent of claim 1and a carrier.
 3. A biocontrol agent comprising Plectosporium tabacinumCL98-103 deposit number PTA-3463 (ATCC).
 4. A composition comprising thebiocontrol agent of claim 3 and a carrier.
 5. The composition of claim2, wherein said carrier comprises clay, alginate, diatomaceous earth,growth medium, or a combination thereof.
 6. The composition of claim 3,wherein said growth medium is selected from the group consisting ofsolid growth medium or liquid growth medium.
 7. The composition of claim4, wherein said growth medium is solid growth medium.
 8. The compositionof claim 7 wherein said solid growth medium is selected from the groupconsisting of potato dextrose agar, Czapek-Dox agar, lima bean agar, V-8juice agar, oatmeal agar, tryptic soy agar, dextrose tryptone agar,Cooke rose bengal agar, prune agar, malt extract agar, syntheticnutrient poor agar, Sabouraud dextrose agar, water agar and cornmealagar.
 9. The composition of claim 7, wherein said growth medium isliquid growth medium.
 10. The composition of claim 9, wherein saidliquid growth medium is selected from the group consisting of V-8 juicemedium, Modified Richard's solution (MSR), Yeast extract broth (YEB),Richard's solution (RS), Czapek-Dox broth (CDB), Trichoderma medium(TM), Tryptic soy broth (TSB), Potato dextrose broth (PDB), Nutrientbroth (NB), Colletotrichum truncatum medium (CTM), Malt extract broth(MEB) or a combination thereof.
 11. A method for the biocontrol of weedscomprising, administering to said weeds an effective amount of thebiocontrol agent of claim
 1. 12. The method of claim 11, wherein saidweeds are cleavers.
 13. The method of claim 12, wherein said cleaverscomprise herbicide-resistant cleavers, herbicide-susceptible cleavers,or a combination thereof.
 14. The method of claim 12, wherein saidbiocontrol agent is administered to said cleavers in conjunction with aherbicide.
 15. The method of claim 11, wherein said biocontrol agent isadministered to said weed at about the one whorl stage or earlier. 16.The method of claim 11, wherein said biocontrol agent comprises asurfactant.
 17. The method of claim 16, wherein said surfactant isSilwet L-77.
 18. The method of claim 17, wherein said surfactant ispresent in an amount of about 0.05% to about 0.1% by volume.
 19. Acomposition comprising spores of P. tabacinum CL98-103 and a carrier.20. A method for the biocontrol of weeds comprising, administering tosaid weeds an effective amount of the composition of claim
 19. 21. Amethod for the biocontrol of weeds comprising, administering aneffective amount of the biocontrol agent of claim 1 to said weeds undernon-aquatic conditions.
 22. A method for the biocontrol of weedscomprising, administering an effective amount of the composition ofclaim 19 to said weeds under non-aquatic conditions.